Inulin is a type of polysaccharides, and is widely distributed throughout the natural world. Colloidal inulin exists in the tubers of plants of the family Asteraceae, such as dahlia, Jerusalem artichoke and Inula japonica or in the root of chicory. Unlike starch, inulin is dissolved in warm water, and has a structure wherein D-fructofuranose is polymerized by dehydration onto the fructose side of sucrose by β-(2→1) linkages. The molecular weight differs depending on the chain length of fructose. Plant-derived inulin can be said to be an aggregate of compounds differing in their molecular weights. The average degree of polymerization ranges from 32 to 34 according to Dictionary of Biological Science (IWANAMI SHOTEN, PUBLISHERS, 2nd edition (1978)), and is about 30 according to Dictionary of Physics and Chemistry (IWANAMI SHOTEN, PUBLISHERS, 3rd edition (1979)). The molecular weight is about 5,000 according to Dictionary of Biochemistry (TOKYO KAGAKU DOZIN CO., LTD., 1st edition (1985)), while according to the literature of W. Praznik (Journal of Chromatography, 348, 187–197 (1985)) the mean molecular weight is 2,282 to 17,000 while the degree of polymerization somewhat varies depending on the types of plants. The molecular size of inulin is limited within a certain range.
Since inulin is dietary fiber that is difficult to digest, it has attracted interest as a dietary fiber. Further, as its effects include, for example, increasing the growth of Bifidohacterium, the demand for inulin is increasing amidst the recent boom in health-consciousness.
Inulin has been mainly produced in areas outside Japan where inulin is produced by cultivating a plant, such as dahlia, chicory, or Jerusalem artichoke, and drying the extracts from the rhizoma, and it is generally consumed as a foodstuff. Inulin is not produced in Japan because commercial cultivation of these plants is difficult.
To acquire inulin in Japan, there is thus no choice but to import. Such imported inulin is more expensive than a domestic substance having functions analogous to inulin. This can be considered as a problem related with industrial use. In addition, the yield of plant-derived inulin depends on the crop conditions, as the raw material of inulin is extracted from the plant. Besides, a problem associated with plant-derived inulin is that the inulin content reduces in value by, for example, autolysis, unless extraction is performed immediately after harvest. Furthermore, in the case of plant-derived inulin, purification is extremely difficult because of varied fructose chain lengths. Thus, currently available plant-derived inulin is commercialized by roughly fractionating as a raw material a solution containing inulin with varied chain lengths, and then drying by spraying. Therefore, there remains a problem that even though the purity as inulin may be high, there is a lack of uniformity in the chain lengths.
On the other hand, the above-mentioned higher plants from which inulin can be extracted obviously contain an enzyme for producing inulin, and it has already been shown by M. Luscher et al. (FEBS Letter 385, 39 (1996)) that inulin is produced from sucrose using an enzyme that is extracted from such a plant. This mechanism is driven by the cooperative action of two types of enzymes: sucrose 1-fructosyltransferase (SST), a sucrose which performs transfer of fructosyl between sucroses, and β-(2→1) fructan 1-fructosyltransferase (FFT), a β-(2→1) fructan which transfers fructose moieties between fructans having a degree of polymerization of 3 or more.
However, it is impractical to employ this mechanism on an industrial scale to prepare a large amount of enzyme from plant bodies, because it is both time- and labor-consuming.
In addition to the plant-derived inulin, a method for producing analogues of inulin by action of microbial enzyme has been reported.
For example, N. Kopeloff et al. reported in 1920 that the conidiospores of Aspergillus sydowi have invertase activity and produce a levan type of fructan from sucrose (J. Biol. Chem., 43, 171 (1920)). Later, J. R. Loewenberg et al. revealed that the polysaccharide has an inulin type conformation having β-(2→1) linkages of fructose (Can. J. Microbiol., 3, 643, (1957)).
Thereafter, G. Kawai et al. reported in 1973 that when conidiospores of Aspergillus sydowi were allowed to react with sucrose, production of polyfructan and oligofructan was observed, and that like higher plant-derived inulin, the polyfructan is in the shape of a straight chain having β-(2→1) linkages, but lacks glucose at its end, and its molecular size is about 20,000,000 which is far greater than that of higher plant-derived inulin (Agric. Biol. Chem., 37, (9), 2111, (1973)).
Then, Nakakuki et al. proposed a method for producing oligofructan and macromolecular fructan by treating sucrose with the cells of Aspergillus sydowi. The produced fructan is a linear polyfructan having glucose at its end and having fructose linked by β-(2→1) linkages. The oligofructan in this case was described as having a degree of polymerization of 5 or less, while the macromolecular fructan has a molecular weight ranging from 1.8×105 to 1.4×107 (JP Patent Publication (Unexamined Application) No. 61-187797).
Harada et al. have also proposed a method for producing polyfructan from sucrose using the conidiospores of Aspergillus sydowi, and describe that the molecular weight of the polyfructan in this case was around 10,000,000 (JP Patent Publication (Unexamined Application) No. 5-308885).
Hidaka et al. have proposed a method for producing linear fructan having β-(2→1) linkages by allowing fructosyltransferase produced by microorganisms belonging to the genus Aspergillus or Fusarium to act on sucrose (JP Patent Publication (Unexamined Application) No. 55-40193). However, the fructan produced in this case is an oligosaccharide wherein 1 to 4 molecules of fructose are bound to sucrose, so that it is defined as a substance different from inulin in molecular size.
Furthermore, Rosell et al. have reported that some of Streptococcus mutans, which is considered a pathogen of dental caries, produce enzyme for producing the analogue of inulin (Acta. Chem. Scand., B28, 589). However, the inulin analogue differs from inulin in that it is a quite giant molecule having the molecular weight of 20,000,000, and has β-(2→6) linkages in a straight chain of β-(2→1) linkages.
As described above, substances that have been so far produced using enzymes derived from microorganisms are referred to as inulin type polyfructan in order to distinguish them from the plant-derived inulin, because the substances have properties largely differing from those of the above described plant-derived inulin (for example, their molecular size is large or they have a different binding format compared to the plant-derived inulin).
Therefore, there has been no established technology to date for producing inulin using enzyme derived from a microorganism.